The invention describes a method for obtaining a glycoprotein composition with increased Man5 and/or afucosylated glycoforms.
Protein glycosylation is one of the most important post-translation modifications associated with eukaryotic proteins. The two major types of glycosylations in eukaryotic cells are N-linked glycosylation, in which glycans are attached to the asparagine of the recognition sequence Asn-X-Thr/Ser, where “X” is any amino acid except proline, and O-linked glycosylation in which glycans are attached to serine or threonine. N-linked glycans are of further two types—high mannose type consisting of two N-acetylglucosamines plus a large number of mannose residues (more than 4), and complex type that contain more than two N-acetylglucosamines plus any number of other types of sugars. In both N- and O-glycosylation, there is usually a range of glycan structures associated with each site (microheterogeneity). Macroheterogeneity results from the fact that not all N-glycan or O-glycan consensus sequences (Asn-X-Ser/Thr for N-glycan and serine or threonine for O-glycan present in the glycoproteins) may actually be glycosylated. This may be a consequence of the competitive action of diverse enzymes during biosynthesis and are key to understanding glycoprotein heterogeneity (Mariño, K., (2010) Nature Chemical Biology 6, 713-723).
The process of N-linked glycosylation begins co-translationally in the Endoplasmic Reticulum (ER) where a complex set of reactions result in the attachment of Glc3NAc2Man9 (3 glucose, 2 N-acetylglucosamine and 9 mannose) to a carrier molecule called dolichol, that is then transferred to the appropriate point on the polypeptide chain (Schwarz, F. and Aebi M., (2011) Current Opinion in Structural Biology, 21:576-582 & Burda, P. & Aebi M., (1999) Biochimica et Biophysica Acta (BBA)General Subjects Volume 1426, Issue 2, Pages 239-257). The glycan complex so formed in the ER lumen is modified by action of enzymes in the Golgi apparatus. If the saccharide is relatively inaccessible, it is likely stay in the original high-mannose form. If it is accessible, then many of the mannose residues may be cleaved off and the saccharide further modified, resulting in the complex type N-glycans structure. In the cis-Golgi, mannosidase-1 may cleave/hydrolyze a high mannose glycan, while further on, fucosyltransferase FUT-8 fucosylates the glycan in the medial-Golgi (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84).
Thus the sugar composition as well as the structural configuration of a glycan structure depends on the protein being glycosylated, the cells/cell lines, the glycosylation machinery in the Endoplasmic Reticulum and the Golgi apparatus, the accessibility of the machinery enzymes to the glycan structure, the order of action of each enzyme and the stage at which the protein is released from the glycosylation machinery.
In addition to the “in vivo” factors listed above, “external factors” may also affect the glycan structure and composition of a protein. These include the conditions in which the cell line expressing the protein is cultured, such as the medium composition, the composition and timing of the feed, osmolality, pH, temperature etc.
Studies by Kaufman et al and Yoon et al show a reduction in protein sialylation upon decrease in temperature (Kaufman, H., Mazur X., Fussenegger, M., Bailey, J. E., (1999) Biotechnol Bioeng. 63, 573-578; Trummer, E., Fauland, K., et. al. (2006) BiotechnolBioeng. 94 1045-1052); Yoon S. K., Song, J. Y., Lee, G. M., (2003) Biotechnol Bioeng. 82: 289-298). Further, reducing temperature can increase overall protein production by prolonging cell viability, which should, in principle, improve glycosylation. (Moore A, Mercer J, Dutina G, Donahue C J, Bauer K D, Mather J P, Etcheverry T, Ryll T. (1997), Cytotechnology. 23:47-54).
Likewise, Borys et al has shown that a deviation from optimum pH results in decrease in the expression rate as well as the extent of glycosylation of proteins (Borys M. C., Linzer, D. I. H., Papoutsakis (1993), BIO/technology 11 720-724). The culture pH of a hybridoma cell line has been shown to affect the resulting galactosylation and sialylation of the monoclonal antibody (Muthing J, Kemminer S E, Conradt H S, Sagi D, Nimtz M, Karst U, Peter-Katalinic J. (2003) Biotechnol Bioeng 83:321-334).
Further, methods for altering glycosylation by culturing cells expressing glycoproteins in cell culture medium comprising metal ions, in particular manganese, have been described. Crowell et al. has shown that the addition of manganese to the cell cultures increased galactosylation which in turn facilitated an increase in O- and N-linked glycosylation (Crowell C K, Grampp G E, Rogers G N, Miller J, Scheinman R I., (2007), Biotechnol Bioeng. 96:538-549).
U.S. Pat. No. 7,972,810 describes a process for increasing the sialylation in hyperglycosylated erythropoietin by addition of manganese in the concentration range of 0.04-40 μM.
Studies by Pacis et al. show a decrease in mannose glycoforms upon supplementing cell culture medium with manganese. In particular glycoforms bearing mannose5 (Man5) glycans decreased from ˜25% to ˜14% in a medium of ˜400 mOsm/Kg and from ˜13% to ˜5% in a medium of ˜300 mOsm/Kg upon supplementation with 1 μM Manganese (Pacis, E., Yu, M., Autsen, J., Bayer, R. and Li, F. (2011), Biotechnology and Bioengineering, 108: 2348-2358. doi: 10.1002/bit.23200). Likewise, US2011/0053223 discloses a cell culture process for accumulation of mannose5 bearing glycoforms by culturing cells in medium comprising manganese at a concentration of 0.25 μM or less.
The structure and composition of the glycan moieties of a glycoprotein can have a profound effect on the safety and efficacy of therapeutic proteins, including its immunogenicity, solubility and half life. Antibodies with high mannose content have become of interest because of the differential clearances of the antibodies bearing Man5 glycans and Man7, 8 or 9 glycans. Studies by Wright and Morrison show faster clearances for antibodies bearing Man7, 8, 9 glycans when compared to Man5 glycans (Wright and Morrison, (1994), J. Exp. Med. 180:1087-1096, 1998, J. Imnnmology, 160:3393-3402). Further, high mannose antibodies that were generated with kifunensine treatment showed higher ADCC activity and greater affinity to FcγRIIIA (Zhou Q. et al., (2008), Biotechnol Bioeng 99(3):652-665). Binding to the FcγIII receptor is dependent on the fucose content of the Fc glycans where a reduction in fucose can increase effector function. Fucose-deficient IgG1 s have shown a significant enhancement of ADCC up to 100-fold (Mori K, (2007), Cytotechnology 55(2-3):109-114. and Shields R L. (2002), J BiolChem 277(30):26733-26740). As afucosyl antibodies have become recognized as having potentially higher therapeutic potency due to enhanced ADCC function, high mannose antibodies, in particular antibody composition with increase Man5 glycan which also lack fucose can be of immense therapeutic benefit.
The present invention describes a process of obtaining an antibody composition comprising an enhanced Man5 and/or afucosylated glycans. Further, the invention describes a method for enhancing Man5 glycans and afucosylated glycans in the antibody composition by culturing cells in a media supplemented with manganese.